Identification of pericytes in periodontal ligament and characterization as mesenchymal stem cells

Abstract

Currently, there is a consensus regarding the biological importance of the mesenchymal stem cells (MSCs) in periodontal defect area upon regeneration of supportive periodontal tissue. It has been suggested that MSCs would be localized in perivascular region of different organs/tissues, having characteristics similar to pericites, which present the ability to migrate and proliferate in cases of tissue damage. However, there is no evidence showing that, cells from periodontal ligament characterized as mesenchymal and undifferentiated correspond to the same cell populations identified as pericites. Besides, there is no evidence regarding these cells distribution in healthy periodontal tissues and damaged by chronic periodontal disease. Thus, this study aims: 1) isolate and characterize phenotypically pericites populations from periodontal ligaments of humans and 2) to evaluate the distribution of pericites/mesenchymal cells in healthy periodontal tissues and damaged by chronic inflammatory periodontal disease. For this purpose, it will be selected 3 adult individuals with indication to third molar extraction to obtain primary cultures of periodontal ligament cells (PDL). From these PDL cells, pericites populations (CD146 positive cells) will be purified by magnetic labeling techinique and characterized towards undifferentiated mesenchymal phenotype (markers expression CD105, CD166, STRO-1 e OCT-4) and endothelial/hematopoietic phenotype (expression of markers CD34 and CD45), through flow citometry and immunohistochemistry techiniques. Then, it will be evaluated the potential of CD146 positive cells to differentiate into osteoblastic and adipogenic phenotype, using the alizarin red-based assay (formation of mineral nodules), oil red O assay (staining of lipidic vacuoles) and gene expression of RUNX-2, ALP, OCN (osteoblastic markers), PPAR³2 and LPL (adipogenic markers) by the quantitative PCR techinique (PCRq). Following, cotton ligatures will be placed in 20 male mouse (Rattusnorvegicus, albinus, Wistar) to periodontal disease induction. After 7 and 21 days of induction, ligatures will be removed and animals sacrificed. Then, it will be performed histometric analysis and immunohistochemistry assay to determine the pattern of distribution of CD105+ STRO-1+ CD146+ in healthy periodontal tissues and damaged by chronic inflammatory periodontal disease. (AU)